Biophysical and structural studies of protein and enzyme mechanism, evolution, and engineering

项目总结/摘要 在生物学研究的几个正在进行的革命中，有两个（可视化日益复杂的能力 生物分子机器和组装以及创造新的蛋白质结构和相应的 功能）是我们实验室研究计划的基础。多年来，我们将其 （i）确定基因靶向蛋白的结构、机制和生物学作用 和碱基修饰酶，以及（ii）采用蛋白质工程来产生新的蛋白质折叠，组装， 和功能，并测试我们对蛋白质形式和功能的理解。我们现在计划更新和扩大 根据我们的研究使命，通过解决核酸领域的几个问题和难题， 酶学、分子识别和蛋白质工程。 前两个项目将建立在我们的经验研究和工程归巢核酸内切酶（也 称为“大核酶”）。在这样做的过程中，我们将回答围绕进化，识别 （i）序列特异性微生物RNA内切核酸酶和（ii）真核生物RNA内切核酸酶的机制和工程化能力 反转录转座子我们计划利用我们对这些系统的基础研究来创造新的工具， 转录组学分析和基因组工程。第三个项目涉及当前的 蛋白质工程的挑战：生物分子界面的精确设计， 立体特异性氢键网络，以促进亲和性，特异性和可逆性的平衡，这是一种 生物分子相互作用和调节的标志。为了做到这一点，我们将创造并结合蛋白质- 蛋白质和蛋白质-小分子的结合功能，通过创建新的合成配体诱导 蛋白质多聚化系统。该项目将有助于分子设计和工程领域， 优化策略的准确设计的立体有择氢键网络，促进 蛋白质-蛋白质和蛋白质-配体识别和结合。

Project Summary/Abstract Among the several ongoing revolutions in biological research, two (the ability to visualize increasingly complex biomolecular machines and assemblages and the ability to create novel protein structures and corresponding functions) represent the foundation of our laboratory's research program. For many years, we have divided its activities between (i) determining the structures, mechanisms and biological roles of gene targeting proteins and base modifying enzymes, and (ii) employing protein engineering to create new protein folds, assemblages, and functions, and to test our understanding of protein form and function. We now plan to renew and expand upon our research mission by address several questions and problems in the fields of nucleic acid enzymology, molecular recognition, and protein engineering. The first two projects will build upon our experience studying and engineering homing endonucleases (also called `meganucleases'). In doing so, we will answer questions surrounding the evolution, recognition mechanisms and engineerability of (i) sequence-specific microbial RNA endonucleases and (ii) eukaryotic retrotransposons. We plan to leverage our basic studies of these systems to create new tools for transcriptomic analyses and genome engineering, respectively. The third project addresses a current challenge in protein engineering: the accurate design of biomolecular interfaces that rely heavily on stereospecific hydrogen-bond networks to facilitate the balance of affinity, specificity and reversibility that is a hallmark of biomolecular interactions and regulation. To do so, we will create and then couple together protein- protein and protein-small molecule binding functions through the creation of novel synthetic ligand-induced protein multimerization systems. The project will contribute to the field of molecular design and engineering by optimizing strategies for the accurate design of stereospecific hydrogen bond networks for that facilitate protein-protein and protein-ligand recognition and binding.